Field of the Invention
The present invention relates to systems and methods for amplifying nucleic acids. In some embodiments, the invention relates to microfluidic PCR analysis systems using microfluidic temperature controlled channels.
Discussion of the Background
The amplification and detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
Several different real-time detection chemistries now exist to indicate the presence of amplified DNA. Most of these depend upon fluorescence indicators that change properties as a result of the PCR process. Among these detection chemistries are DNA binding dyes (such as SYBR® Green) that increase fluorescence efficiency upon binding to double stranded DNA. Other real-time detection chemistries utilize Forster resonance energy transfer (FRET), a phenomenon by which the fluorescence efficiency of a dye is strongly dependent on its proximity to another light absorbing moiety or quencher. These dyes and quenchers are typically attached to a DNA sequence-specific probe or primer. Among the FRET-based detection chemistries are hydrolysis probes and conformation probes. Hydrolysis probes (such as the TaqMan® probe) use the polymerase enzyme to cleave a reporter dye molecule from a quencher dye molecule attached to an oligonucleotide probe. Conformation probes (such as molecular beacons) utilize a dye attached to an oligonucleotide, whose fluorescence emission changes upon the conformational change of the oligonucleotide hybridizing to the target DNA.
A number of commercial instruments exist that perform real-time PCR. Examples of available instruments include the Applied Biosystems PRISM 7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0. The sample containers for these instruments are closed tubes which typically require at least a 10 μl volume of sample solution.
More recently, a number of high throughput approaches to performing PCR and other amplification reactions have been developed, e.g., involving amplification reactions in microfluidic devices, as well as methods for detecting and analyzing amplified nucleic acids in or on the devices. Thermal cycling of the sample for amplification is usually accomplished in one of two methods. In the first method, the sample solution is loaded into the device and the temperature is cycled in time, much like a conventional PCR instrument. In the second method, the sample solution is pumped continuously through spatially varying temperature zones.
To have good yield of a target product, one has to control the sample temperature at different levels very accurately. And to reduce the process time, one has to heat up or cool down the sample to desired temperature very quickly.
One specific approach for regulating temperature within the devices is to employ external temperature control sources. Examples of such sources include, but are not limited to, heating blocks and water baths. Another option is to utilize a heating element such as a resistive heater that can be adjusted to a particular temperature. Another temperature controller includes Peltier controllers (e.g., INB Products thermoelectric module model INB-2-(11-4)1.5). This controller can be utilized to achieve effective thermal cycling or to maintain isothermal incubations at any particular temperature.
In some devices and applications, heat exchangers can also be utilized in conjunction with one of the temperature control sources to regulate temperature. Such heat exchangers typically are made from various thermally conductive materials (e.g., various metals and ceramic materials) and are designed to present a relatively large external surface area to the adjacent region. Often this is accomplished by incorporating fins, spines, ribs and other related structures into the heat exchanger. Other structures include coils and sintered structures. In certain devices, heat exchangers such as these are incorporated into a holding space, chamber or detection area.
Conventional heat exchangers that can be utilized in certain applications are discussed, for example, in U.S. Pat. No. 6,171,850 which discloses a reaction receptacle that includes a plurality of reservoirs disposed in the surface of a substrate. Additional methods of temperature control for microfluidic systems are known which include, for example: a thermal cycling system using the circulation of temperature controlled water to the underside of a microtiter plate (U.S. Pat. No. 5,508,197); a thermal cycling system using infrared heating and air cooling (U.S. Pat. No. 6,413,766); a microfluidic chip where flow travels through several static temperature zones (U.S. Pat. No. 6,960,437); the use of exothermic and endothermic materials to heat up and cool down the PCR samples (U.S. patent application publication US2005/012982).
In conventional systems temperature accuracy and thermal cycling speeds are issues to be resolved. For example, the accuracy of the temperature of any bath used to heat a microchannel and the bath's subsequent conduction of heat to the microchannel is important in that certain stages of PCR processing take place at well-defined temperatures. The thermal cycling speed refers to the time between stabilization from one temperature to another in a heating cycle. For example in the PCR process, the thermal cycling speed refers to the time to shift from 95° C. to 55° C. to 72° C. The faster the thermal cycling speeds and the more accurate the temperature stabilization, the more efficient PCR processes can be performed.
There is a need for improved systems and methods for amplifying nucleic acids and for systems and methods for microfluidic thermal control.